Isolates of Cryptosporidium from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.
A total of 430 avian-derived fecal specimens were randomly collected from selected Western Australian commercial aviaries, poultry farms, hatcheries, wildlife parks, and the Perth Zoo and screened for the presence of Cryptosporidium by PCR. Of these, 27 Cryptosporidium-positive isolates were detected, characterized, and compared with 11 avian-derived isolates from the Czech Republic at the 18S rRNA and actin gene loci. Sequence and phylogenetic analysis identified four genetically distinct genotypes, avian genotypes I to IV, from various avian hosts. In addition, the host range for Cryptosporidium galli was extended. Cryptosporidium muris and Cryptosporidium andersoni were also identified in a tawny frogmouth and a quail-crested wood partridge, respectively.Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged, and wild birds (33), and the parasite has been reported in more than 30 avian species worldwide, belonging to orders Anseriformes, Charadriiformes, Columbiformes, Galliformes, Passeriformes, Psittaciformes, and Struthiniformes (5,6,9,10,13,14,15,16,17,20,22,23,28,31,32,35,38,39). However, few studies have examined the genetic diversity of Cryptosporidium sp. among avian hosts.There are currently three accepted avian species of Cryptosporidium, C. baileyi, C. meleagridis, and C. galli, based on biological and genetic differences (3,24,25,26,29). Of these, only C. meleagridis is known to infect humans (37). Recent studies have identified six novel avian genotypes: goose genotypes I and II, the duck genotype, two unnamed genotypes in Canada geese (Branta canadensis) (9, 39), and the Eurasian woodcock (Scolopax rusticola) genotype (30). In addition to the host-specific avian genotypes identified, C. hominis and C. parvum were also identified in Canada geese (39). However, this was thought to be due to mechanical transmission (8,39).These studies indicate that the extent of genetic diversity among avian-derived Cryptosporidium isolates is greater than previously thought and that this warrants further investigation. The high prevalence of Cryptosporidium in domesticated, caged, and wild birds potentially represents a risk to humans (e.g., pet owners and poultry farmers) and other mammals that become infected with the parasite. The purpose of this study was to examine the prevalence and host range of Cryptosporidium species and genotypes among caged, domesticated, and wild birds and to determine if they are potential reservoirs of human-infectious species of Cryptosporidium. MATERIALS AND METHODSFecal sample collection and DNA extraction. A total of 430 fresh fecal samples of various avian host species were randomly collected from March 2004 to December 2004 at selected commercial aviaries, poultry farms, hatcheries, wildlife parks, and the Perth Zoo. All samples were collected into individual 250-ml fecal collection pots and stored at 4°C until required. An additional 11 avianderived fecal specimens from the Czech Republic that were positive by microscopy for Cryptosporidium...
ABSTRACT:Cryptosporidium galli Pavlasek, 1999, described from the feces of birds, is redescribed with additional molecular and biological data. Oocysts are ellipsoidal, are passed fully sporulated, lack sporocysts, and measure 8.25 X 6.3 p[m (range 8.0-8.5 X 6.2-6.4 pVm) with a length-width ratio of 1.30 (n= 50). Oocysts are structurally similar to those of Cryptosporidium baileyi described from chickens, but in addition to being considerably larger than oocysts of C. baileyi, these oocysts infect the proventriculus in a variety of birds and not the respiratory tract. Oocysts were successfully transmitted from chickens to chickens, and morphologically similar oocysts also were observed in a variety of exotic and wild birds (Order Passeriformes, Phasianidae, Fringillidae, and Icteridae). Molecular and phylogenetic analyses at the 18S rRNA, HSP70, and actin gene loci demonstrate that this species is genetically distinct from all known species and genotypes of Cryptosporidium and, thus, was named C. galli. Cryptosporidial infections MATERIALS AND METHODS OocystsOocysts of Cryptosporidium were obtained from the feces of spontaneously infected birds (see Table I) from the Czech Republic. Fecal samples were examined and oocysts purified using routine coprological methods (Breza, 1957; Pavlasek, 1991). Oocysts were stored in 2.5% potassium dichromate at +4 C until required for molecular analysis. For each isolate, 50 oocysts were measured at X 1,000 magnification. The lengths, widths, and shape index of oocysts of C. baileyi (n= 50), C. meleagridis (n= 40), and C. galli (pooled values of isolate Czech-B 1-25 and Czech B1-31, n= 100) were compared using an analysis of variance. Values were considered statistically different if P < 0.05. Transmission studiesOocyst inocula were prepared by washing purified oocysts with phosphate-buffered saline (pH 7.2) to remove potassium dichromate. The number of oocysts fed to birds was determined by counting on a hemocytometer. Two 9-day-old chickens and two 40-day-old chickens were each fed 3 X 104 oocysts. Feces were examined daily for >60 days for oocysts using routine coprological methods (Breza, 1957; Paylasek, 1991).Deoxyribonucleic acid extraction, polymerase chain reaction, and sequence analyses Oocysts were purified and deoxyribonucleic acid (DNA) was extract-
The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.
Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris ; (1) C. muris genotype A ; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.
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